Implantable biosensor and methods of use thereof

ABSTRACT

Provided herein is a stabilized oxygen transport matrix that includes a reversible oxygen binding protein, such as hemoglobin, immobilized throughout the stabilized oxygen transport matrix. The stabilized oxygen transport matrix is used to transport oxygen and can be used as an oxygen transport region and a reaction region of an analyte sensor, such as an implantable glucose sensor. The reversible binding protein can also function as an oxygen probe within the analyte sensor.

RELATED APPLICATION DATA

This application claims the benefit of priority under 35 U.S.C.§119(e)(1) of U.S. Ser. No. 60/531,447, filed Dec. 18, 2003, the entirecontent of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to glucose monitoring and morespecifically to implantable glucose sensors.

2. Background Information

Diabetes is a disease of insufficient blood glucose regulation. Innon-diabetic people, the body's beta cells monitor glucose and deliverjust the right amount of insulin on a minute-by-minute basis for tissuesin the body to uptake the right amount of glucose, keeping blood glucoseat healthy levels. In diabetics this healthy regulation system primarilyfails due to the following two factors, either alone or in combination:

1) insufficient insulin production and secretion, 2) a lack of normalsensitivity to insulin by the tissues of the body.

The first major breakthrough in treating diabetes was the discovery ofinsulin. The backbone of today's treatments relies on this discovery,and the patient's self-initiative and compliance. Two types of diabetesmellitus are common. Type 1 diabetes accounts for 5-10% of all cases,and Type 2 diabetes accounts for 90-95% of the diabetic population. InType 1 diabetes, the disease requires insulin injections to maintainlife, in addition it requires healthy eating and exercise. Treating type2 diabetes may require insulin, but the disease may be controllable withoral medication, weight loss, a careful diet and a regular exerciseprogram.

There is still no magic pill to treat diabetes. Current drugs have thepotential to eliminate complications altogether, if only the patientknew when and how much to take. A program of very frequent sampling isrequired to provide both the rate and extent of glycemic excursions.This set of glucose measurements is absolutely necessary information tocalculate the timing and amount of corrective actions needed toeffectively treat Diabetes and prevent complications. The importance ofblood glucose monitoring has been underscored by the results of theDiabetes Control and Complications Trial, which showed that many of thelong-term complications of diabetes could be prevented by close bloodglucose regulation.

However, current blood glucose tests are painful, requiring fingersticking to obtain a blood sample. They are inconvenient due todisruption of daily life and difficult to perform in long-term diabeticpatients due to calluses on the fingers and poor circulation. Withpresent technology, the average diabetic patient tests his/her bloodglucose levels less than twice a day versus the recommended 4-7 timesper day. Further, even the recommended testing schedule is far fromsufficient to allow blood glucose normalization.

Thus with present technology, the necessary monitoring is frequentlyunachieved chore. The required sampling schedule cannot realistically beexpected of even the most committed patients during the day and is notfeasible at night. Present blood glucose monitoring methods are notautomatic, chronically requiring user initiative. This system cannottherefore be relied upon to detect spontaneous hypoglycemia or otherglycemic excursions. Consequently, even the most diligent patients failto avoid severe complications. As a result $85 billion was spent in 2002on treating Diabetes complications, including loss of sight, loss ofkidney function, loss of limbs, vascular disease, heart failure, stroke,coma, and severe constant pain.

New glucose monitoring methods are needed to address these shortcomingsAn automatic, painless, and convenient means of continuous glucosemonitoring could provide the information needed for adequate control.This would greatly reduce the complications seen in these patients andthe associated health care costs of their treatment.

In order to meet the needs of continuous glucose monitoring fordiabetes, the monitoring process must satisfy the following:

Require no sample preparation (the measurements occur automatically)

-   -   Be highly selective and sensitive    -   Provide a rapid response to changes in glucose    -   Provide highly repeatable/reproducible measurements    -   Operate with stability and low drift

A number of different technologies have been applied to develop aglucose sensor to meet these needs. However, the most direct route tobring a successful device to the market is to develop a disposablesensor that operates in the subcutaneous tissue. This minimizes the riskof serious complications associated with a fully implanted device.

A very successful method that satisfied all of the above requirementsfor biosensing is enzyme based ampermetric electrode sensing. Thismethod was intended to operate in a homogenous oxygen environment withhigh oxygen availability, such as a major blood vessel in the body. Theemployed method consumed oxygen, effectively maintaining a zero oxygenconcentration at the electrode surface in order to measure oxygen.However, this approach is not directly applicable to the subcutaneoustissue.

As is often the case, the type of sensing method applied will impact theability to achieve success in new sensing environments. Many sensingmethods will perform well under in vitro or carefully controlledconditions, but will then fail to perform well in the body. Theirfailure has been attributed to inadequate selectivity, electrodepoisoning, and insufficient glucose sensitivity.

High glucose selectivity is essential to provide an accurate measurementof glucose in the body. The selectivity of a measurement refers to thedegree to which a particular analyte may be determined in a complexmixture without interference from other constituents in the mixture. Inthe body, there is a complex mixture that may be termed the tissuematrix in which glucose must be measured. The tissue matrix containsmany constituents that are constantly changing and which may interferewith varying types of measurement approaches. The constant state of fluxof the tissue matrix prevents a calibration from being established forselective measurement through a technique such as multiple regression toremove the impact of unmeasured interfering constituents of the matrix.

A full range of approaches from non-invasive to invasive are beingdeveloped in an attempt to bring a new kind of glucose sensor to market.However, while appealing, non-invasive optical measurements aregenerally not sufficiently selective for glucose without a detailedknowledge of the matrix being probed. The optical measurement isperformed by focusing a beam of energy onto the body. The energy ismodified by the tissue after transmission through the target area. Asignature of the tissue content is produced by the energy exiting thetissue. The energy leaving is a function of chemical componentsencountered as well as thickness, color and structure of the tissuematrix through which the energy passes. In the body, the tissue matrixis constantly changing. Additionally, constant changes in the externalenvironment, and their impact on the skin provide a non-stationaryenvironment. This poses a severe challenge for purely opticalmeasurements to be highly selective for glucose.

To achieve sufficient selectivity for glucose, the enzyme glucoseoxidase may be employed in a semi-invasive approach. Clark and Lyonsfirst used the strategy of combining the specificity of a biologicalsystem to achieve the necessary selectivity for glucose measurements ina tissue matrix. Glucose oxidase has a high specificity for glucose.This enzyme reduces glucose to gluconic acid and peroxide in thepresence of oxygen and water.

By coupling glucose oxidase with a suitable transducer, glucoseconcentration may be measured by monitoring either the production ofperoxide or the consumption of oxygen.

However, problems exist in the direct application of both of theseapproaches. Hydrogen peroxide probes often suffer from electrochemicalinterference by oxidizable species in a complex matrix such asencountered in the body. The electrode oxidizes these otherelectroactive constituents as well as hydrogen peroxide, which resultsin measurements with a net positive and variable error. The hydrogenperoxide if not eliminated may also have an undesirable reaction withthe surrounding tissue as well as degrade the oxidase enzyme necessaryfor the operation of the sensor over the course of sensor operation.Additionally, unless oxygen is available in excess to the glucose beingreduced by the reaction, variations in bulk oxygen will also change howmuch glucose is oxidized, resulting in erroneous measurements if theoxygen concentration influencing the reaction is not directly accountedfor in a calibration, or prevented from impacting reaction dynamics. Theproblem of sensitivity to varying oxygen concentration is also presentin approaches based on measuring oxygen consumption. Unfortunately, thesubcutaneous tissue environment has been shown to have both a variableand heterogeneous oxygen concentration. These measurements musttherefore address background oxygen variations to accurately determinethe amount of oxygen being consumed, necessary for accurate glucosemeasurement.

Thus, to effectively utilize glucose oxidase a series of problems shouldbe overcome. First, sufficient oxygen should be available for thereaction to proceed. Second, regardless of how glucose oxidase iscoupled to a transducer, if oxygen is not available in excess, as willbe the case in the subcutaneous tissue, then knowledge of oxygenconcentration is needed to provide a completely selective measurement ofglucose. Third, glucose oxidase should be coupled to a detector in amanner satisfying the requirements of biosensing stated above.

Much work has been done on coupling glucose oxidase to an electrode.However, problems of stability, drift, selectivity, and sensitivity mustbe overcome if using an electrode system. Extensive efforts have beendevoted for stabilizing the electrode, minimizing the error ofelectroactive interference, and preventing “electrode poisoning”;however, an alternative approach is to avoid using an electrode as atransducer by selecting an optical measurement method. In thesubcutaneous environment, oxygen is scarce, making an optical methodthat measures the result of the glucose oxidation reaction while notconsuming oxygen even more desirable.

In coupling glucose oxidase with an optical transducer, an additionalproblem of selective sensing of oxygen is posed. Fortunately, opticalmeans of selectively sensing oxygen are well established.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery of astabilized oxygen transport matrix that includes a reversible oxygenbinding protein, such as hemoglobin, immobilized throughout thestabilized oxygen transport matrix. The stabilized matrix collectsoxygen, stabilizes oxygen dynamics, and rapidly transports oxygen withinthe matrix. For example, the stabilized matrix can transport oxygen froma region of relatively high oxygen surrounding one surface of thestabilized matrix, to a region of lower or no oxygen on a secondsurface. Furthermore, the stabilized matrix can transport oxygen to asite within the matrix or juxtaposed to the matrix, where a reactionoccurs in which oxygen is consumed.

The stabilized oxygen matrix provides numerous functions including rapidtransport of oxygen in an implantable sensor and rapid transport ofoxygen to an artificial tissue. Accordingly, provided herein in oneembodiment is a method for transporting oxygen from a first area havinga relatively high oxygen concentration to a second area having arelatively low oxygen concentration, that includes contacting oxygenfrom the first area, to a first surface of a stabilized oxygen transportmatrix that includes a reversible oxygen binding protein immobilizedthroughout the stabilized oxygen transport matrix, and transportingoxygen away from the first surface of the stabilized oxygen transportmatrix to a second surface of the oxygen transport matrix, wherein thesecond surface of the oxygen transport matrix contacts the second area.

In illustrative embodiments, the present invention is based, at least inpart, on the discovery that the stabilized oxygen transport matrix canbe used to rapidly transport oxygen in a biosensor, especially animplantable glucose sensor, to overcome challenges of implantablesensors, such as spatial and temporal changes in oxygen concentration atthe site of an implant. Additionally, the present invention is based, atleast in part, on the discovery that a substantially non-oxygenconsuming probe can be coupled with a selective mediator such as glucoseoxidase with appropriately defined geometry and boundary conditions todevelop a glucose sensor that will operate accurately under low oxygentension such as typical in the subcutaneous tissues. The reversibleoxygen-binding protein transporter can be used to increase the supply ofoxygen to the glucose oxidase reaction site in a more homogenous manner,providing an environment for the reaction to proceed that is moresuitable for making sensitive glucose measurements with a broad dynamicrange. Furthermore, a reversible oxygen binding protein, or anadditional oxygen-sensitive dye, can be used to optically measurechanges in oxygen concentration that result from the glucose-oxidasecatalyzed reaction of glucose that is being mediated by oxygen. Bychoosing this approach, the issues of selectivity are overcome when areference oxygen measurement is taken at a region in the glucose sensorthat is distant from a site where glucose enters the sensor, but havingan oxygen concentration (at the reference region) that provides ameasure of the oxygen concentration that would have been present at thesite where glucose enters the sensor in the absence of glucose.

In one embodiment, the present invention provides an enzymatic-basedsensor capable of selectively and sensitively monitoring glucose in thesubcutaneous tissues under low oxygen tensions. The sensor includes anoxygen transport region comprising a first reversible oxygen bindingprotein, an oxygen permeable first surface in communication with anexternal environment, and an oxygen permeable second surface which isimpermeable to the target analyte; a target analyte reaction region incommunication with the oxygen transport region at the oxygen permeablesecond surface, wherein the target analyte reaction region comprises atarget analyte oxidase enzyme, and a target analyte-permeable surface;and a sensing region comprising at least one detector probe incommunication with the target analyte reaction region. Typically, atarget analyte and oxygen impermeable surface is located in the sensorsuch that the sensing region is in between the target analyte and oxygenimpermeable surface and the oxygen transport region.

The target analyte can be, for example, glucose, galactose, lactose,peroxide, cholesterol, amino acids, alcohol, or lactic acid. In certainillustrative examples, the target analyte is glucose and the sensor is aglucose sensor. For example, the glucose sensor can be an implantableglucose sensor such as a transcutaneous glucose sensor.

The first reversible oxygen binding protein can be an engineeredhemeprotein or a heme derivative. In illustrative examples, the firstreversible oxygen binding protein is myoglobin or hemoglobin.

The sensing region can further include an oxygen probe. For example, theoxygen probe can be a second reversible oxygen binding protein, such asan engineered hemeprotein or a heme derivative. In illustrativeexamples, the first and the second reversible oxygen binding protein ishemoglobin.

In certain exemplary sensors provided herein, at least one substantiallynon-oxygen consuming detector probe is used. The detector probe canfurther include a spectrometer. At least one detector probe, in certainexamples, is capable of emitting and/or receiving light at an oxygensensitive engineered hemeprotein, or heme derivative absorptionwavelength. For example, one or more emitters can emit light toward oneor more receivers, and the emitters and the receivers can both enter thesensor through a first end. The one or more emitters can be one or morefiber optic fibers that are formed into a loop such that light exitingfrom the emitter fiber optic fibers travels in a path that issubstantially opposite to light from a light source that enters theemitter fiber optic fibers.

In another embodiment, provided herein is a method for measuring aconcentration of an analyte, comprising transporting oxygen from anexternal environment through an oxygen transport region within a sensorto an analyte reaction region of the sensor using a first reversibleoxygen binding protein; reacting a portion of the transported oxygenwith the analyte enzymatically in the analyte reaction region to form aproduct; and measuring oxygen or a product of the reaction of oxygen andthe analyte in a sensing region comprising the analyte reaction regionor a sensing region in contact with the analyte reaction region, therebymeasuring the concentration of the analyte.

In illustrative aspects, the analyte being measured is glucose,galactose, lactose, peroxide, cholesterol, amino acids, alcohol, orlactic acid. In a particularly illustrative embodiment, the analytebeing measured is glucose. The measuring can be performed repeatedly,for example at least at a first time point and a second time point,thereby providing information regarding changes in analyte concentrationover time. In certain examples, oxygen is transported from the externalenvironment comprising subcutaneous tissue to the analyte reactionregion using an engineered hemeprotein or a heme derivative as the firstreversible oxygen binding protein. For example, oxygen is transportedfrom the external environment to the analyte reaction region usinghemoglobin as the first reversible oxygen binding protein.

Oxygen can be measured, for example, by measuring oxygen binding of anoxygen probe. Oxygen binding can be measured using a spectrometermeasuring absorption of the oxygen binding protein such as a secondreversible binding protein. The second reversible oxygen binding proteincan be an engineered hemeprotein or a heme derivative, such ashemoglobin. In illustrative aspects, hemoglobin is both the firstreversibly binding protein and the second reversible binding protein.

The measuring is typically performed using one or more detector probes,for example a population of substantially non-oxygen consuming detectorprobes, emitting light towards one or more non-oxygen consumingreceivers at an oxygen sensitive engineered hemeprotein or hemederivative wavelength. In certain aspects, light travels through one ormore emitter fiber optic fibers from a light source emitting in a firstdirection, and then travels in a second direction that is substantiallyopposite the first direction through at least a portion of the sensingregion where it is received by one or more receiver fiber optic fibers.In a related aspect, light travels through one or more emitter fiberoptic fibers from a light source emitting in a first direction throughat least a portion of the sensing region, and then travels in a seconddirection that is substantially opposite the first direction where it isreceived by one or more receiver fiber optic fibers. In other words theemitter fibers or the receivers can include a loop in the fiber.

The method can include a reference measurement within the sensingregion. Typically, the analyte enters the reaction region through ananalyte inlet and the analyte concentration is determined by using thesignal from at least one oxygen probe that is sufficiently close to theanalyte inlet to be sensitive to analyte-derived oxygen gradients withinthe sensing region along with the signal from at least a second oxygenprobe that is sufficiently far from the analyte inlet to act as areference probe for the oxygen profile. The reference probe can besufficiently far from the analyte inlet so that the signal from thereference probe is not substantially affected by the analyte enteringthe analyte inlet.

In illustrative aspects, a spatially substantially uniform oxygenconcentration is present at the boundary of the reaction region and theoxygen transport region. Furthermore, the oxygen transport regiontypically provides a sufficient oxygen flux to the reaction region suchthat the enzyme reaction is not oxygen limited. In addition, the size ofthe analyte inlet, the concentration of an enzyme in the reactionregion, and the concentration of the reversible oxygen binding proteinin the reaction region can be tuned to provide an analyte sensitiveoxygen gradient near the inlet such that the desired dynamic range ofanalyte concentrations can be measured, and to provide a analyteinsensitive oxygen reference concentration distal to the inlet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic drawing of glucose sensor geometry accordingto illustrative aspects of the present invention.

FIG. 2 is a plot of photodiode voltage corresponding to diode lasertransmission through a Hemoglobin (Hb) film bound in an oxygenimpermeable membrane save for a small region at one end exposed to itssurroundings. The signal represents the changing Hb absorption at 635 nmlaser light. In the time preceding reference ‘A’ room air was applied atthe exposed region of the Hb film. At reference ‘A’, a 5% oxygen gasmixture was applied to the exposed region of the Hb film, followed byroom air again applied to the exposed region of the Hb film at reference‘B’.

FIG. 3A provides a schematic drawing of an illustrative example of aglucose sensor of the present invention, as disclosed in Example 2. FIG.3B provides a schematic diagram of the illustrative glucose sensor ofExample 2 connected to a light emitter 490, light detector 495, andsignal processing 498 unit.

FIG. 4A provides a schematic drawing illustrating the path of fiberoptic bundles and light in an illustrative glucose sensor of the presentinvention. FIG. 4B provides a schematic drawing illustrating fiber opticbundles and light emission in an illustrative glucose sensor of thepresent invention that includes a closer view of the coupling region 425where the detection bundle 415 receives light from the emitter bundle410. FIG. 4C provides a schematic drawing of a cross-sectional view of adetection bundle (also called a receiver bundle) 415 and an emitterbundle 410. In FIG. 4B, separation is illustrated between the emitterbundle and the reaction region. However, in other aspects of theinvention, the emitter bundle is in contact with, and butted up against,the reaction region.

FIGS. 5A and 5B provide a schematic diagram and graphs illustratingexpected oxygen and glucose concentration changes in a glucose sensoraccording to the present invention. The upper graph in FIGS. 5A and 5Brepresents expected spatial fluctuations in oxygen concentration insubcutaneous tissue outside the glucose sensor at the surface whereoxygen enters the oxygen transport region 001 of the glucose sensor. Thelower graph in FIG. 5A illustrates expected oxygen concentrations acrossthe oxygen injector surface 6 and within the glucose reaction zone inthe absence of glucose. The lower graph in FIG. 5B illustrates expectedspatial changes in oxygen concentration (dashed line) and glucoseconcentration (solid line) across the glucose reaction zone 2 at a planewithin the glucose reaction zone 2 after entry of glucose through aglucose inlet 7.

FIG. 6 illustrates detection of glucose in a stabilized glucoseoxidase-hemoglobin thin matrix The results are presented as plots ofglucose concentrations within a water bath with a 5% O₂+95% N₂ gasmixture bubble in, plotted against the corresponding photodiode voltagewhen the reaction gel was interrogated at a location of 50 um or 100 umfrom the glucose inlet 7.

FIG. 7 shows the calibrated response of the glucose oxidase-hemoglobinthin gel of FIG. 6 plotted against the TrueTrack™ glucose metermeasuring the same glucose solutions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thata reversible oxygen-binding protein, such as hemoglobin, can be used asan oxygen conduit in a stabilized oxygen transport matrix. Thestabilized matrix collects oxygen, stabilizes oxygen dynamics, andrapidly transports oxygen away from a region of relatively high oxygensurrounding one surface of the stabilized matrix, to a region of loweror no oxygen on a second surface. The stabilized oxygen matrix providesnumerous functions including rapid transport of oxygen in an implantablesensor and rapid transport of oxygen to an artificial tissue.

Accordingly, provided herein are biosensors, especially implantableglucose sensors, that utilize the stabilized oxygen transport matrix asan oxygen transport region and a reaction region to overcome challengesof implantable sensors, such as spatial and temporal changes in oxygenconcentration at the site of an implant. The reversible oxygen-bindingprotein conduit can be used to increase the supply of oxygen to aselective mediator such as glucose oxidase in a homogenous, measurablemanner. Furthermore, the oxygen binding protein within a stabilizedoxygen transport matrix can be used to optically measure changes inoxygen concentration within regions of the matrix that result from thereactions within the matrix in which oxygen is consumed, such as aglucose-oxidase catalyzed reaction of glucose with oxygen.

In a first embodiment, the present invention provides a sensor thatincludes an oxygen transport region comprising a first reversible oxygenbinding protein, an oxygen permeable first surface in communication withan external environment, and an oxygen permeable second surface which isimpermeable to the target analyte; a target analyte reaction region incommunication with the oxygen transport region at the oxygen permeablesecond surface, wherein the target analyte reaction region comprises atarget analyte oxidase enzyme, and a target analyte-permeable surfaceand a sensing region comprising at least one detector probe incommunication with the target analyte reaction region. Typically, atarget analyte and oxygen impermeable surface is located in the sensorsuch that the sensing region is in between the target analyte and oxygenimpermeable surface and the oxygen transport region. The sensing regionis typically in optical, electrical, or molecular communication with thetarget analyte reaction region.

In certain aspects, the sensor includes an oxygen transport means fortransporting oxygen from a surface in contact with an externalenvironment to a target analyte reaction region and a detection meansfor detecting and/or measuring oxygen, the target analyte, or a productof the reaction of oxygen and the target analyte within a sensing regionwithin the reaction region or contacting the reaction region. Numerousexamples of the oxygen transport means and detection means are providedherein.

The first reversible oxygen binding protein can be, for example, anengineered hemeprotein or a heme derivative. In illustrative examples,the first reversible oxygen binding protein is myoglobin, or in furtherillustrative examples, hemoglobin.

Many different target analytes can be detected and measured by thesensors provided herein provided that the target analyte reacts withoxygen. For example, the target analyte can be galactose, lactose,peroxide, cholesterol, amino acids, alcohol, or lactic acid. In anillustrative example, the target analyte is glucose and the sensor is aglucose sensor. It will be understood that the teachings provided hereinwith respect to glucose can be used to make and use sensors for otheranalytes that react with oxygen.

Accordingly, as illustrated in FIG. 1, provided herein is a glucosesensor 100 that includes an oxygen transport region 001, that includes areversible oxygen binding protein, an oxygen permeable first surface 004and an optional oxygen permeable surface 005 in communication with anexternal environment, and an oxygen permeable second surface 006,sometimes referred to herein as the oxygen injector 006; a glucosereaction region 002 in mass transport communication with the oxygentransport region 001 at the oxygen permeable second surface, wherein theglucose reaction region includes, the reversible oxygen binding protein,a glucose oxidase enzyme and a glucose permeable surface 007, sometimesreferred to herein as the glucose inlet 007, and a sensing region 003 inoptical or electrical communication with the glucose reaction region002, wherein the sensing region 003 includes a probe, such as an oxygenprobe, a glucose probe, or a probe that binds to, or is otherwiseaffected by, a product of the reaction of oxygen and glucose. Thesensing region 003 can be a region within the reaction region 002, or aregion sufficiently in contact with the glucose reaction region torepresent the spatial and temporal profile of glucose or other targetanalyte, oxygen, and/or reaction product within the reaction region,that is interrogated by one or more detector probes 009, which areindividual sensing elements 009 within a sensing interface. Furthermore,the device can have an axis of symmetry 010. The oxygen transport region001 is an exemplary stabilized oxygen transport matrix of the presentinvention. Communication between the oxygen transport region 001 and theglucose reaction region 002 typically occurs across a surface 006 wherethe oxygen transport region 001 and glucose reaction region 002 are incontact. The communication between the oxygen transport region 001 andthe glucose reaction region 002 can be any type of communication thatinvolves diffusion and permits the transport of oxygen from the oxygentransport region 001 into the glucose reaction region 002. Thecommunication can be, for example, the movement of liquid, gas, and/orions from the oxygen transport region 001 to the glucose reaction region002.

In certain aspects of a sensor of the present invention, an outermembrane 011 can surround all or a portion of the device. The membraneis typically permeable to oxygen and glucose, but impermeable to atleast some biomolecules such as proteins, especially biomolecules suchas proteins within the sensor and of immune proteins. Therefore, themembrane has a cutoff, for example, of 1 kDa, 2 kDa, 5 kDa, 10 kDa, or25. In some aspects, the membrane pores can be up to about 10 um, or themembrane can be laminar with an outer layer with pores up to 10 um tofacilitate cell infiltration, and the inner layer can have pore sizes ofas small as 10 kDa to prevent protein transport.

In another embodiment, provided herein is a method for measuring aconcentration of an analyte, that includes transporting oxygen from anexternal environment through an oxygen transport region within a sensorto an analyte reaction region of the sensor using a first reversibleoxygen binding protein; reacting a portion of the transported oxygenwith the analyte enzymatically in the analyte reaction region to form aproduct; and measuring oxygen or a product of the reaction of oxygen andthe analyte. The measuring is performed in a sensing region thatincludes the analyte reaction region or a sensing region in contact withthe analyte reaction region. As discussed herein, virtually any analytecan be detected and measured using the methods herein provided that theanalyte reacts with oxygen. In certain aspects, the analyte beingmeasured is galactose, lactose, peroxide, cholesterol, amino acids,alcohol, or lactic acid. In illustrative aspects, the analyte beingmeasured is glucose. It will be understood that the teachings withrespect to measuring glucose can be applied to methods for determiningvirtually any analyte that reacts with oxygen.

Accordingly, provided herein is a method for measuring glucoseconcentration, that includes transporting oxygen from an externalenvironment through an oxygen transport region 001 to a glucose reactionregion 002 using a reversible oxygen binding protein, reacting a portionof the transported oxygen with glucose in the glucose reaction region002 to form a product; and measuring oxygen, glucose, or a product ofthe reaction of oxygen and glucose in a sensing region 003 within theglucose reaction region 002 or in contact with the glucose reactionregion 002, thereby measuring glucose concentration. In illustrativeexamples, oxygen is measured in the reaction region to measure glucoseconcentration.

In an illustrative aspect, a glucose sensor 100 provided herein is animplantable glucose sensor, for example a transcutaneous glucose sensor.Typically, transcutaneous glucose sensors can be removed by a user. Somesensors provided herein can be delivered as part of, or connected to acatheter, into a blood vessel. The methods provided herein measurerelative or absolute concentrations of glucose in tissue, bytransporting oxygen from tissue in the region around the sensor to theglucose reaction zone using an oxygen transport means. In illustrativeaspects, the implantable sensor is implanted in subcutaneous tissue andoxygen is transported by the oxygen transporter to the reaction zonefrom tissue surrounding at least a portion of one side of the oxygentransporter, and typically all sides of the transporter except for theside that abuts the glucose reaction zone. In embodiments where thesensor is implanted in subcutaneous tissue, the sensor can be asubcutaneous sensor, but typically the sensor is a transcutaneous sensorin which a portion of the sensor traverses the skin of an animal, forexample a mammal, such as a human. For example, probes connected to thesensor can traverse the skin to reach a light source and a detector thatare located outside the body. In subcutaneous embodiments, the entiresensor as well as the detector can be located subcutaneously. The lightsource can be located subcutaneously, or an external light source can beused that is not physically connected to the sensor. The sensor can alsobe used to measure glucose concentrations as well in the vasculature,bodily excretions, or other tissues such as muscle, or fat, that mayhave higher or lower vascularization and oxygen tension thansubcutaneous tissue.

The method and sensors provided herein can be used to monitor changes inabsolute or relative glucose concentration. For example, diabetics canuse the sensors and methods to monitor their glucose levels to determinewhether changes in glucose concentrations are being adequately managedby a treatment regime. Furthermore, diabetics can use the sensor 100 inthe control of insulin delivery. Therefore, methods provided hereintypically include at least measuring glucose concentrations at a firsttime point and a second time point, thereby providing informationregarding changes in glucose concentration over time. The devices andmethods provided herein can be used to measure relative or absolutechanges in glucose concentrations, with high sensitivity and precisionacross the entire range of at least 10 to 600 mg/dl which brackets theclinically relevant concentrations of 70 mg/dl and less which definehypoglycemia, and 250-400 mg/dl and up which define hyperglycemia. Thedevice probes the spatial distribution of oxygen in a glucose reactionzone in which changes of oxygen concentration are used to infer changesof glucose concentration according to the kinetics of the glucoseoxidase enzyme. Sensor calibration, which may include the oxygenreference measurement described herein, allows absolute glucoseconcentration measurements based on changes in the oxygen concentrationdistribution. Patients can be alerted by the sensor if their bloodglucose falls below factory or user set concentrations, for example 90mg/dl and/or if rates of glucose concentration decrease exceeds factoryor user set rates, for example 10 mg/dl*min⁻¹ over a sustained period.

In certain illustrative examples, the sensors and methods providedherein are used to measure glucose concentrations on an ongoing basis,such as periodically at repeated time points (e.g., first, second,third, fourth, fifth, sixth, seventh, etc. time points) or continuously.Glucose measurements can be taken at virtually any interval, forexample, in real time for continuous monitoring, or at intervals of lessthan a second, seconds, minutes, hours, or days. Continuous monitoringincludes monitoring using pulsed light that is emitted into the sensingregion at a relatively high frequency (e.g., once every 5 or 10 secondswith 1 to 5 second duration, or every one second with a 0.1-0.5 secondduration). The sensors can be easily inserted beneath the skin, forexample by simply holding the sensor housing, which may be a cylinder of1 inch diameter and ½ inch thickness from which a needle protrudeswithin which is the sensor, and driving the needle through the skin intothe subcutaneous tissue. Alternatively, the housing can be loaded into aspring-loaded device which is placed against the skin, and uponactuation of a lever or button, said device can insert the needle underthe skin. The device can then remain in its transcutaneous orsubcutaneous location for a period of hours, days, weeks, months, oreven years, to allow periodic or continuous sensing of blood glucoselevels.

A biosensor 001 according to the present invention typically utilizes asystem designed around a reaction between a compound and oxygen that ismediated by an oxidase enzyme. As a specific embodiment, the enzymeglucose oxidase is used to catalyze the reaction of oxidizing glucose togluconic acid and peroxide in the presence of water and oxygen.

C₆H₁₂O₆+O₂+H₂O→C₆H₁₂O₇+H₂O₂  (1)

This reaction consumes both the desired analyte (glucose) and oxygen andcreates products other than oxygen and the desired analyte. Thisreaction is dependent on both the analyte and the oxygen present at thecatalytic site of the enzyme. Thus a glucose concentration is coupled toan oxygen reduction and new product concentrations. The sensor can thenmeasure the concentration of glucose by measuring the reduction in theconcentration of oxygen, glucose, or a product of the reaction of oxygenand glucose, as it is consumed or produced in the oxidase enzymemediated reaction. This typically requires that a background oxygenmeasurement at the site of the glucose reaction be provided to interpretchanges in the oxygen field.

Illustrative examples of biosensors provided herein are intended tooperate under physiological tissue conditions, in which the availabilityof oxygen is significantly less than the availability of glucose.Intravenous sensor placement proximal to the heart has shown to be anexcellent environment for long term glucose sensing in terms of oxygenavailability and very limited rejection by the body. However, there aremortal risks associated with the intravenous placement making anon-vascular placement desirable since it is less invasive and does notpose some of the potential life threatening risks of the vascularimplant site. Yet in subcutaneous tissues it has been shown that thetissue oxygen tension is quite low in comparison to atmospheric orarterial oxygen tension. Many tissues of the human body have an oxygentension equivalent to between about 5% oxygen in nitrogen or lower, andthere may be a ratio of glucose to oxygen sometimes as high as 100 to 1in subcutaneous interstitial fluids.

As the glucose oxidation reaction (equation 1) requires equal molaramounts of oxygen and glucose, the reaction may be limited by a lack ofoxygen. If bulk oxygen is considerably less available than bulk glucose(which is the norm in the subcutaneous environment), then theavailability of oxygen to the catalytic site will govern the reactionrather than the availability of glucose. Under these conditions, thereaction chamber of the sensor is like a car engine that had thecarburetor flooded with fuel, the combustion reaction in the engine willnot proceed efficiently with too much fuel and not enough oxygen. Thecombustion reaction will still proceed but only to the extent that thereis oxygen available as there is excess fuel. This limits the sensitivityof the sensing system to glucose.

To overcome the physical limitations of glucose, oxygen, and enzymeworking within a simple unregulated solution, biosensors according tothe present invention are constructed much like an engine. Within thisanalogy, the fuel is glucose, the spark plug is the catalytic site ofthe enzyme, and the oxygen is necessary for the reaction to proceed.This creates a transport and reaction problem. The goal is to get thebalanced amounts of oxygen and glucose to the catalytic site of theenzyme. This will make the reaction at the enzyme responsive to changesin the glucose concentration, like a car engine is responsive to changesin the fuel flow controlled by the driver, and not to changes is ambientoxygen. Specifically, the biosensor is designed to have the reaction besensitive to variations in the bulk glucose concentration andinsensitive to transient variations in the bulk oxygen concentration.

The invention overcomes the issue of limited glucose sensitivity byimplementing the necessary boundary conditions for the glucose oxidasereaction to readily occur. This is accomplished by rapidly transportingthe needed oxygen to a reaction region 002 where the boundary conditionsgoverning the glucose reaction are carefully regulated.

The reaction region 002 is formed from multiple boundaries (FIG. 1):

A boundary 006, also referred to herein as the oxygen injector, in thereaction chamber permitting high oxygen transport but no glucosetransport.

A boundary 007, also referred to herein as the glucose inlet, permittingthe entrance of glucose from the bulk into the reaction chamberjuxtapose to the boundary 006. The boundary 007 may also allow oxygen toenter. The entire boundary 007 may be permeable to glucose or maycontain an inlet or inlets that are permeable to glucose.

A boundary 008, called the sensing surface, that is impermeable to bothglucose and oxygen allows sensing of the reaction chamber and is axiallyoffset from the oxygen injector 006 and juxtaposed to the glucose inlet007.

In addition to the three boundaries 006, 007, 008 delineating thereaction chamber, the complete sensor system contains an oxygentransport region 001 and a sensing region 003 contiguous with boundary008. In certain aspects, the reaction region 002 is interrogated with atemperature probe. Readings from the temperature probe can be used in adetermination of absolute concentration of glucose, due to thetemperature dependence of the glucose oxidation reaction. Thetemperature probe can be an optical probe whereby infrared light iscollected by a fiber optic or fiber optic bundle coupled to apyroelectric detector located in the same housing as, and communicatingwith a signal processing unit. The temperature probe can be used as aninput to the calibration for glucose concentration. It can communicate,for example, electronically with a signal processing unit.

The oxygen transport region 001 is designed to collect oxygen, stabilizeoxygen dynamics, and rapidly transport oxygen from an extended regionaway from the reaction chamber to the oxygen injection surface, namedthe oxygen injector, 006, of the reaction chamber. Therefore, the oxygentransport region functions as an oxygen transport means. The oxygentransport region 001 provides a sufficient oxygen flux to the reactionregion 002 such that the enzyme reaction is not oxygen limited. Thesensing element optically interrogates the reaction chamber orsubregion(s) of the reaction chamber to measure oxygen through boundary008. FIG. 5A demonstrates the function of the oxygen transport region001 whereby oxygen from a heterogeneous substantially non-zero oxygenfield outside the first surface of the oxygen transport region istransported to the oxygen transport second surface 006 where it entersthe reaction region 002 as a smoothed oxygen field as is represented bythe graphical plots of oxygen concentration profiles ‘[O₂] outside’, and‘[O₂] sensing surface’ (for the sake of illustration, the sensingsurface 008 and surface 006 will have sufficiently equivalent profilesin the absence of glucose).

A “reversible oxygen binding protein” is a oxygen-carrying protein thatreversibly binds oxygen under physiologically relevant oxygenconcentrations. The reversible oxygen binding protein binds oxygen atsome higher partial pressures and releases oxygen when oxygenconcentrations fall, the change in the saturation of the protein as afunction of the oxygen partial pressure being described by an oxygensaturation (also termed dissociation) curve. For example, the oxygensaturation curves of the reversibly oxygen binding proteins hemoglobin(haemoglobin) or myoglobin have very different loading and unloadingcharacteristics as summarized by their strikingly different oxygensaturation curves. The hemoglobin saturation curve is sigmoidal, whichreflects its cooperative binding, whereas that for myoglobin ishyperbolic which reflects noncooperative binding. Comparing the curvesfor hemoglobin to myoglobin, the saturation of myoglobin is alwayshigher than hemoglobin at all partial pressures, indicating the higheraffinity of myoglobin for oxygen than that of hemoglobin for oxygen.Consequently, in blood capillaries (partial pressure of oxygen is approx20 mmHg) hemoglobin will release its oxygen to the tissues and allowingthe myoglobin to store the oxygen for later releasing it to the tissues.

The reversible oxygen binding protein contains an oxygen binding sitethat is modulated by at least a peptide unit. For example, a heme groupis modulated in hemoglobin or myoglobin. However, the reversible oxygenbinding site may not employ a heme group as is the case for somehemoglobin orthologs. For example, the reversible oxygen bindingproteins found in invertebrates such as hemocyanin found in mollusks andcrabs arthropods which has two Cu ions for the reversible oxygen bindingsite, or Hemerythrin found in some marine invertebrate that also doesnot contain a heme group. The oxygen binding protein may also besynthetically derived with multiple functions such as the recombinanthuman serum albumin (rHSA) incorporating the synthetic heme“albumin-heme” to form an oxygen-carrying plasma protein (J Biomed MaterRes. 2004 Dec. 15; 71A(4):644-51), or such as the recombinant, humananti-sickling beta-globin polypeptide designated beta(AS3)(betaGly(16)-->Asp/betaGlu(22)-->Ala/betaThr(87)-->Gln) was designed toincrease affinity for alpha-globin (J Biol. Chem. 2004 Jun. 25;279(26):27518-24. Epub 2004 Apr. 14). Additionally, other geneticchanges may be used to achieve different variants of reversible oxygenbinding proteins, such as the Escherichia coli produced recombinanthemoglobin(alpha 29leueine->phenylalanine, alpha 96valine-->tryptophan,beta 108asparagine-->lysine) which exhibits low oxygen affinity and highcooperativity combined with resistance to autoxidation (Biochemistry.1999 Oct. 5; 38(40):13433-42.

The reversible oxygen binding protein in illustrative examples ispresent in the glucose reaction region (also called the glucose reactionzone) and/or the sensing region, as well as the oxygen transport region.The reversible oxygen binding protein is present in the oxygen transportregion typically at a concentration of 0.1 g/dl-100 g/dl, for example7.5 g/dl. The reversible oxygen binding protein can be increased orreduced in concentration until such a point at which the extraordinaryoxygen transport rates, as illustrated in the Examples, are no longerachievable. The upper limit of the concentration of the reversibleoxygen binding protein is also limited by the maximum ability to loadthe protein based on the mass of the protein.

The reversible oxygen binding protein in illustrative embodiments is anaturally occurring oxygen carrier, for example a heme derivative or anon-heme containing oxygen binding protein, or the reversible oxygenbinding protein is an engineered hemeprotein. An engineered hemeproteinis a non-naturally occurring heme-based oxygen carrier or a modifiednaturally occurring heme-based oxygen carrier protein suitable forperforming the function of the reversible oxygen-binding proteindisclosed herein. The protein is typically engineered using recombinantDNA technologies. A heme derivative is a naturally occurring heme-basedoxygen carrier such as an oxygen binding protein, which includes, forexample, hemoglobin and myoglobin, or naturally occurring orthologs andvariants thereof. In certain illustrative examples, the reversibleoxygen binding protein is hemoglobin. The reversible oxygen bindingprotein typically undergoes relatively large changes in O₂ saturationwith respect to small changes in O₂ partial pressure. For example, intranscutaneous sensor applications, the reversible oxygen protein canundergo relatively large changes in saturation in O₂ partial pressuresup to at least, for example, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10%,illustrative partial pressures in certain subcutaneous tissues.Accordingly, in methods provided herein oxygen is transported from theexternal environment to the glucose reaction zone using hemoglobin,myoglobin or an engineered heme-protein as the reversible oxygen bindingprotein. In illustrative embodiments, the reversible oxygen bindingprotein has a near linear saturation at oxygen pressures expected forthe application of the sensor. For example, the reversible oxygenbinding protein can have a near linear saturation response in O₂ partialpressures between 0 and 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10%, intranscutaneous sensors. In certain particularly illustrative examples,the reversible oxygen binding protein is hemoglobin at a concentrationof between 1 and 25 g/dl, for example between 5 and 10 g/dl, or in anillustrative example at 7-8 g/dl, and even more particularly for exampleat 7.5 g/dl. Variants of hemoglobin are known within and betweenspecies. Furthermore, hemoglobin or engineered heme-proteins (Winslow R.M., .MP4, a new nonvasoactive polyethylene glycol-hemoglobin conjugate.Artif Organs. 2004 September; 28(9):800-6; Komatsu T, et al.,Physicochemical characterization of cross-linked human serum albumindimer and its synthetic heme hybrid as an oxygen carrier. BiochimBiophys Acta. 2004 Nov. 18; 1675 (1-3):21-31)) can be designed andproduced using approaches such as recombinant DNA techniques (Leon R G,et al., High-level production of recombinant sulfide-reactive hemoglobinI from Lucina pectinata in Escherichia coli High yields of fullyfunctional holoprotein synthesis in the BLi5 E. coli strain. ProteinExpr Purif. 2004 December; 38(2):184-195) that may have normal oraltered oxygen binding properties that are ideal for the biosensorsprovided herein, depending on the specific application of the biosensor.Additionally, the hemoglobin or engineered hemeprotein can be modifiedto attain different oxygen saturation curves suitable for delivery orimproved measurement properties for a transcutaneous sensor operating atthe expected low physiological oxygen tensions. Examples ofmodifications include crosslinking as part of the process to create thestabilized matrix with hemoglobin using gluteraldehyde or other agentsto shift the p50 of the hemoglobin saturation curve. In the case ofgluteraldehyde modification, the extent of the p50 shift will bedependent on the source of hemoglobin (e.g., human or bovine), the molarratio of gluteraldehyde to hemoglobin and whether additional cofactorsare included during crosslinking (Keipert, P. E., et al., Functionalproperties of a new crosslinked hemoglobin designed for use as a redcell substitute. Transfusion. 1989 November-December; 29(9):768-73; andEike, J. H., Effect of glutaraldehyde concentration on the physicalproperties of polymerized hemoglobin-based oxygen carriers. BiotechnolProg. 2004 July-August; 20(4):1225-32) In certain aspects, the oxygentransport region includes a series of bands each comprising a differentengineered hemeprotein, myoglobin, hemoglobin variant, or hemoglobin, orcombination thereof, with an altered oxygen saturation curve, such thatreversible oxygen binding proteins in each band have different oxygenloading and/or unloading characteristics. Such a design can be used tofine tune the glucose sensor for a particular application.

It will be understood that although the sensor and methods providedherein are illustrated using the detection of glucose, the teachingsherein can be used in sensors and methods for detecting other analytesas well. Accordingly, provided herein are methods and sensor devices formeasuring an analyte, wherein the device includes an oxygen diffusionregion that includes a reversible oxygen binding protein at aconcentration sufficient to transport oxygen from one or more oxygenentry surfaces of the oxygen diffusion region to an analyte reactionzone that includes an oxidase enzyme that catalyzes the reaction ofoxygen with the analyte. The device typically includes a sensor regionfor measuring the analyte, oxygen, or a product catalyzed by the oxidaseenzyme. The device in illustrative examples, measures oxygen bymeasuring changes in absorption of the reversible oxygen binding protein(i.e. utilizes the reversible oxygen binding protein as both an oxygentransporter and an oxygen probe). Virtually any analyte can be measuredusing the inventive sensors and methods where oxygen can be used as aco-factor for an enzymatic reaction involving the analyte, and O₂ is notpresent upon completion of the enzymatic reaction. In addition toglucose oxidase, as illustrated herein, other analytes that can bemeasured include galactose, using galactose oxidase, lactose, usinglactose oxidase, peroxides, using peroxidases. cholesterol usingcholesterol oxidase, amino acids using amino acid oxidase, alcohol usingalcohol oxidase, and lactic acid using lactate oxidase.

The characteristics of a reversible oxygen binding protein can beanalyzed with respect to the illustrative reversible oxygen bindingprotein hemoglobin. The nonlinear loading and unloading characteristicsof hemoglobin and myoglobin greatly facilitate the transport of oxygenfrom the bulk through the oxygen transport region 001 and into thereaction chamber via the oxygen injector 006. The transportcharacteristics of hemoglobin move the oxygen through the oxygentransport region 001 to the low oxygen tension regions at a speed far inexcess of normal diffusion. This extremely rapid transit coupled withthe unique loading and unloading characteristics of hemoglobin create aspatially substantially uniform or self-consistent distributionidentifiable by the oxygen reference value across the profile of theoxygen transport region 001 at the injector surface 006. Additionally,these unique loading and unloading characteristics buffer variations inthe oxygen supply being transported through the oxygen transport region001. A value at the reference should map to the same oxygen distributionregardless of the distribution outside the first surface of the oxygentransport region. Here ‘map’ is intended to mean that a reference valuecodes for only one oxygen distribution across the injector. While anonspatially uniform injector oxygen field or profile may result in anonmonotonic oxygen field in the glucose reaction zone which may becompensated for in an appropriate calibration, a spatially substantiallyuniform injector oxygen field or profile provides a simpler oxygen toglucose calibration. Therefore, the oxygen transport region 001 providesa temporally substantially damped oxygen concentration profile withrespect to bulk oxygen dynamics at the boundary 006 of the glucosereaction zone 002 and the oxygen transport region 001. These uniquecharacteristics allow the conduit 001 to stretch far into the bulktissue environment, effectively uniting a supply of oxygen fromcapillaries and arterioles to the reaction region 002 where it is neededfor the enzyme mediated oxidative reaction. The extensive oxygencollecting area of the oxygen transport region, 001 moves oxygen to thesmall volume of the reaction region 002 supplying ample oxygen for theglucose oxidase reaction. In this sense, the oxygen transport region 001acts as an artificial microcirculation which couples the reaction region002 to the microcirculation of the tissue.

The oxygen conducting potential of the oxygen transport region 001 alsoameliorates another key roadblock in short term subcutaneous sensing.Numerous attempts have been made to make subcutaneously implanteddevices for measuring glucose in diabetics. However, experimentaldevices have ultimately failed due to a lack of sensitivity to glucoseafter implantation. The lack of sensitivity has been ascribed to amongother issues, the isolation of the biosensor electrode by layers ofscar-like tissue. The scale and biocompatible composition of theexterior of the oxygen transport region 001 should allow it to remainproximal to arterioles and numerous capillaries. This should allowsufficient oxygen to remain transported to the sensing site even if thedevice is encapsulated. Accordingly, the implantable glucose sensor 100of the present invention in certain illustrative embodiments, maycontain a vast array of different geometrical arrangements of the keysensor components which will yield a functioning device. In certainaspects the geometry can be, for example, a cylinder no more than 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 mm³ in total volume. Forexample, in embodiments where the implantable glucose sensor iscylindrically shaped, the diameter of the implantable glucose sensor incertain aspects is no more than 3.4 mm, 0.64 mm, or 0.31 mm. In certainembodiments the diameter of the implantable glucose sensor is thediameter of a 18, 23, or 30 gauge hypodermic needle. In an illustrativeembodiment, the diameter can be as small as 0.030 mm and as large as0.31 or 0.64 mm where the smallest represents the implantation of asingle fiber optic probe and the largest represents fiber optic bundlesimplanted in a syringe needle ranging in gauge from 30 to 23. The lengthof the implantable glucose sensor can be, for example, between 1 mm and100 mm, or in certain preferred illustrative examples, it is between 2mm and 10 mm where length is a trade off between oxygen collection andcomfort.

Accordingly, the lengths of the various boundaries and regions with thesensor provided herein are small enough to permit the sensor to have theoverall total dimensions provided above. For example, the oxygeninjector 006 can be, for example, between 0.05 and 10 mm, 0.1 and 1 mm,or between 0.1 and 0.3 mm in length. The glucose inlet 007 is typicallybetween 0.0001 and 1 mm, or in illustrative examples, between 0.001 and0.1 mm in length. The sensing surface 008 can be, for example, between0.05 and 10 mm in length, and in illustrative examples is between 0.1and 0.3 mm in length. The ratio of the length of the oxygen injector 006to the length of the glucose inlet 7, is typically between 1 and 1000,and in an illustrative embodiment, is between 5 and 100, or morespecifically between 6 and 30. However, it will be recognized that thebiosensors provided herein are scalable and may take on various shapes.The glucose inlet is small enough to assure that a subregion within thereaction region 002 has no detectable glucose. In certain aspects, acharacteristic length of the glucose inlet, defined as the square rootof its area, is no more than ½ of a characteristic length of the glucosereaction region where the characteristic length is defined as thedistance from the glucose inlet to the effective boundary of the glucosereaction region in a direction pointing along the surface normal of theglucose inlet. With respect to ratios of other regions of the sensor ofthe present invention, the length of the oxygen injector 006 can be, forexample, approximately identical, or identical to the length of thesensing surface 008. The ratio of the volume of the oxygen transportregion 001 and the volume of the reaction region 002 is typically atleast 2:1, and in illustrative aspects is 5:1, 10:1, 100:1, 250:1,500:1, or 1000:1. In preferred embodiment ratios of at least 5:1, and ashigh as 200:1 may be implemented where 200:1 represents a 0.050 mm thickreaction region 002 with a 10 mm oxygen transport region 001.

The oxygen transport region 001 in certain examples has across-sectional area of between 0.005 and 1 mm², or in an illustrativeembodiment is between 0.015 and 0.08 mm², the inner diameter crosssectional areas of the 30 and 23 gauge needle respectively, and theglucose reaction region 002 typically has a matching cross sectionalarea along the axis of the oxygen transport region 001, as does thesensing region 003, in certain aspects. The sensing region in certainexamples, has a slightly reduced cross-sectional area compared to theoxygen transport region. In aspects where the glucose inlet 7 is acircular hole, the diameter can be between 1 um and 1 mm, but it will beunderstood that the length of the glucose inlet 7 regardless of shapecan be as large as the distance from the oxygen injector 006 to thesensing surface 008. The glucose reaction region 002 typically has amatching cross sectional area along the axis of the glucose inlet 007 ofbetween about 10 um and 1 mm, for example between 25 um and 250 um, orin certain illustrative examples about 100 um. Not to be limited bytheory, it is believed that the large ratios between cross-sectionalareas along the axis of the oxygen transport region 001 and along theaxis of the glucose inlet 007 (and of the glucose inlet itself), accountfor at least part of the sensor's ability to operate in low oxygenenvironments.

Within the reaction region 002, also referred to as a reaction zone or areaction chamber, a means for rapidly transporting oxygen from theoxygen transport region boundary (i.e. oxygen injector) 006 into thereaction region and to the catalytic sites of the enzymes in thereaction region is provided. This can be accomplished again by employinga reversible oxygen binding protein such as hemoglobin, myoglobin, or anengineered hemeprotein to decrease the resistance to oxygen transportfrom the oxygen injector surface into the catalytic sites of the glucoseoxidase enzyme. Additionally, the presence of a reversible oxygenbinding protein within the reaction region will also decrease thediffusivity of glucose, helping to regulate the reaction by decreasingthe flow of glucose down the established gradient in the reaction region002, as discussed in more detail below. The net result is that theenzymatic reaction remains responsive to changes in bulk glucoseconcentration over a broad range of bulk glucose concentrations, andthat the reaction couples bulk changes in glucose concentration tochanges in the oxygen field in the reaction zone.

In certain aspects, the reversible oxygen binding protein within theoxygen transport region 001, and/or the reversible oxygen bindingprotein and the glucose oxidase in the glucose reaction region 002 canbe placed in a stabilized emulsion to ensure high availability of theoxygen carriers to the catalytic sites of the enzyme in the reactionzone. For example, the stabilized emulsion can be a stabilized matrix.

The teachings herein with respect to an oxygen transport region 001 anda reaction region 002 of a biosensor 100, are applicable more generallyto any method or device for rapidly transporting oxygen from a region ofrelatively high oxygen partial pressure to a region of lower partialpressures, as illustrated in the Examples provided herein. This generalapplicability is especially true for aspects of the invention where theoxygen transport region and/or the reaction region are stabilizedmatrices. The transport typically occurs without convection.Accordingly, provided herein in another embodiment, is a stabilizedoxygen transport matrix that includes a reversible oxygen bindingprotein immobilized in the stabilized oxygen transport matrix. Thereversible oxygen binding protein is typically stabilized throughout thematrix. However, as indicated herein, the concentration of thereversible oxygen binding protein can change within the matrix.

The stabilized matrix collects oxygen, stabilizes oxygen dynamics, andrapidly transports oxygen within the matrix. For example, the stabilizedmatrix can transport oxygen from a region of relatively high oxygensurrounding one surface of the stabilized matrix, to a region of loweror no oxygen on a second surface. Furthermore, the stabilized matrix cantransport oxygen to a site within the matrix or juxtaposed to thematrix, where a reaction occurs in which oxygen is consumed, asillustrated by the reaction region 002 in the glucose sensors providedherein. In illustrative embodiments, the reversible oxygen protein, asindicated herein for other embodiments of the invention, is hemoglobin,myoglobin, or an engineered hemeprotein.

The stabilized oxygen transport matrix can take on virtually any shapeor form provided that at least 1 surface of the oxygen transport regionis permeable to oxygen and either a second surface is permeable tooxygen or oxygen is consumed at a region within the stabilized oxygentransport matrix. For example, the matrix can be one or a series ofsheets or cylinders.

The stabilized oxygen matrix can include a variety of components inaddition to the reversible oxygen binding protein, as disclosed hereinwith respect to an oxygen transport region 001 and a reaction region 002of a sensor 100. For example, the stabilized matrix can include acarrier protein to protect protein function during matrix stabilizationand during sensor operation. The carrier protein in certain illustrativeembodiments is serum albumin and/or gelatin. It has been reported thatadding the carrier protein at 1 to 15% by weight of final concentrationwill protect protein function (U.S. Pat. No. 6,815,186), especially forrelatively low enzyme loadings, typically equal to, or less than, 70% byfinal concentration. In certain aspects, the loading can be as high as15 g/L to 25 g/L at final concentrations. Stabilization of the matrixcan be achieved, for non-limiting example, through crosslinking by atleast one of the following agents: an aldehyde such as glutaraldehyde orformalin, a carbodiimide, an imidoester, a pyrocarbonate, an epoxide orN-hydroxysuccinimid ester. In certain aspects, the stabilized matrixcontains the enzyme catalase or peroxidases in order to decrease theperoxide levels within the matrix. In certain aspects the catalaseloading can be 1 to 20 units/ml (See e.g., glucose oxidase formulationsavailable from Biozyme, United Kingdom) or 100 to 1000 units/mil; andU.S. Pub. Pat. App. No. 2002/0006634). Furthermore, the stabilizedoxygen matrix can include polyglycolic acid (PGA), polylactic acid(PLA), poly(lactic-co-glycolic acid) (PLGA), PLLA, poly(caprolactone)(PCL), poly(dioxanone) (PDS), or neovascularization promoting-fibrouscapsule inhibiting material such as polytetrafluoroethylene (PTFE),expanded polytetrafluoroethylene (ePTFE), polypropylene, and polyvinylalchohol (PVA). These components can be used to stabilize reversibleoxygen binding protein within the oxygen transport matrix. Thereversible oxygen binding protein concentration can be as little as 10%by mass within the polymer, or in certain examples usingpolyethyleneglycol as the polymer, on the order of 5 to 200 partsreversible oxygen binding protein to polymer, and still retain itsoxygen carrying characteristics (See e.g., Wettstein et al,“Resuscitation with polyethylene glycol-modified human hemoglobinimproves microcirculatory blood flow and tissue oxygenation afterhemorrhagic shock in awake hamsters”, Crit. Care Med 2003 Vol. 31, No.6; Moore, “Blood Substitutes: The Future Is Now”, J Am Coll Surg., Vol.196, No. 1, January 2003 and U.S. Pat. No. 5,585,484), incorporatedherein in their entirety by reference. The stabilized oxygen matrix caninclude silicon or can be covered with a coating of silicon, rubber, ora polymer. Furthermore, in certain aspects, all or a portion of thestabilized oxygen transport matrix can be encapsulated by a membrane,such as a non-natural membrane made of, for example, a hydrophobicmaterial such as silastic. In certain aspects, the stabilized oxygentransport matrix includes a reversible oxygen binding proteinimmobilized in the stabilized oxygen transport matrix within the poresof a permeable membrane. The membrane can be made virtually any type ofmembrane material, but in certain aspects, it is a biocompatiblemembrane that can be implanted in an animal, especially a mammal such asa human. In certain aspects the membrane material can be a naturallyderived material such as, but not limited to, collagen, alginate,agarose, hyaluronic acid derivatives, chitosan, fibrin glue, or asynthetic polymer such as polyglycolic acid (PGA), polylactic acid(PLA), poly(lactic-co-glycolic acid) (PLGA), PLLA, poly(caprolactone)(PCL), poly(dioxanone) (PDS), or neovascularization promoting-fibrouscapsule inhibiting material such as polytetrafluoroethylene (PTFE),expanded polytetrafluoroethylene (ePTFE), polypropylene, and polyvinylalcohol (PVA).

In a related embodiment, provided herein is a method for transportingoxygen from a first area having a relatively high oxygen concentrationto a second area having a relatively low oxygen concentration, thatincludes contacting oxygen from the first area, to a first surface of astabilized oxygen transport matrix that includes a reversible oxygenbinding protein immobilized throughout the stabilized oxygen transportmatrix, and transporting oxygen away from the first surface of thestabilized oxygen transport matrix to a second surface of the oxygentransport matrix, wherein the second surface of the oxygen transportmatrix contacts the second area. The reversible oxygen transport matrixis an example of an oxygen transport means. Transport of oxygen from anoxygen transport region 001 to a reaction region 002 of a sensor 100, isan illustrative example of this embodiment.

In yet another related embodiment, provided herein is a method fortransporting oxygen from a first area having oxygen to a reaction area,that includes contacting oxygen from the first area, to a first surfaceof a stabilized oxygen transport matrix that includes a reversibleoxygen binding protein immobilized throughout the stabilized oxygentransport matrix, and transporting oxygen away from the first surface tothe reaction area, wherein oxygen is consumed in a reaction at thereaction area. Transport of oxygen within a reaction region 002 of asensor 100 from a site of relatively high oxygen concentration away froma glucose inlet to a site of glucose closer to the glucose inlet, is anillustrative example of this embodiment. Methods of the presentinvention can be used to transport oxygen over distances from between0.001 mm and 20 mm, for example between 0.01 mm and 0.5 mm,

The stabilized oxygen matrices provide numerous functions including notonly rapid transport of oxygen in an implantable sensor, but also rapidtransport of oxygen to an artificial tissue. As such, the stabilizedoxygen transport matrices provided herein can be used as oxygen deliverysystems for artificial organs or cell/tissue transplants. The stabilizedoxygen matrices are important to achieve desired responses of thetransplant such as, but not limited to, viability, differentiation, andfunction, depends upon oxygen availability. In fact, one or morestabilized oxygen transport matrices can be part of an artificialmicrovasculature, a specific type of oxygen transport region used tocollect oxygen from regions of high partial pressures distal from animplant and unloading the oxygen to the implant where oxygen partialpressure is lower. In these embodiments, the stabilized oxygen transportmatrices can form a spider-like assembly of oxygen transport regions ora reticulated meshwork of matrices, in order to maximum the surface areaof the stabilized oxygen transport matrix that contacts tissue regionsthat have oxygen.

Artificial microvasculatures composed of stabilized oxygen transportmatrices provided herein can also be used to deliver oxygen toendogenous tissues, for non-limiting example, in a disease state, intreatment of cardiovascular disease, in wound healing, in treatment ofcerebral hypoxia or hypoxic encephalopathy, or in limb reattachmentafter amputation. Other applications include supplying oxygen to tumorsto prevent hypoxic-mediated radio and chemo resistance, the treatment ofmedical conditions linked to hypoxia such as rheumatoid and other typesof arthritis, chronic inflammatory bowel disease, skin conditions suchas eczema and psoriasis, diabetic inflammatory vasculopathy and diabeticneuropathy, and tendon degeneration.

Biosensors of the present invention, in certain illustrative aspects,utilize the discovery that an optical method can be coupled with aselective mediator such as glucose oxidase to develop a glucose sensorthat will operate accurately in the subcutaneous tissues. Theoxygen-sensing component of the biosensor is achieved by interrogatingthe oxygen field within the reaction region 002. This is accomplishedelectrically or optically, in which volumes of the reaction region 002bound by the oxygen transport region boundary 006 and the sensingboundary 008 are sampled by one or more detector probes. FIG. 5Billustrates the transformation of the oxygen profile from a relativelyuniform profile as shown in FIG. 5A in the absence of glucose, to asubstantially non-uniform profile upon the entry of glucose through theinlet. As is illustrated in the figure, glucose at the inlet reacts withthe oxygen, as mediated by the enzyme glucose oxidase, whereby oxygen isconsumed. The oxygen consumption determines an oxygen profile within thereaction region which is detectable within the sensing region, which isdependent upon the concentration of glucose, the enzyme loading, and theoxygen flux at the injector surface (for any given device geometry ofthis invention). By design, the oxygen profile distal to the glucoseinlet is sensitive to the injector oxygen concentration and not to theglucose concentration and can be used as an input to map the oxygenprofile to a given glucose concentration. The detector probes, inillustrative aspects, are a series of substantially non-oxygen consumingprobes which in certain aspects will consume less that 10% of the oxygenwithin the sensing region per characteristic time in which thecharacteristic time is the 95% rise time for the oxygen concentrationwithin the sensing region following a step change of oxygenconcentration at its boundary with the reaction region, or in certainaspects, with the injector surface. In other aspects, the characteristictime may be defined as the 95% rise time of oxygen concentration withinthe sensing region following a step change of glucose concentration atthe glucose inlet. The detector probes typically are light conduits thatcollect light from one end and efficiently guide it to a second end.Emitters are detector probes that emit the light that interacts with anoxygen-sensitive media within the reaction region 002. Receiversrecollect light that contacts the oxygen-sensitive media, for analysis.Therefore, emitters and receivers form a detection means. For example,the detector probes can be fiber optic fibers, fiber optic bundles, orlight guides including liquid light guides. However, the detector probesin certain aspects, are substantially non-oxygen consuming electrodesthat measure oxygen or a product of the reaction through a polarographicsensing system. In other aspects, one or more ampermetric probes areused as detector probes to measure oxygen or a product of the reactionof oxygen and an analyte such as glucose, using an ampermetric method.The ampermetric method typically does not consume oxygen but consumes aproduct of the enzyme reaction between the target analyte and oxygen. Incertain aspects, the one or more detector probes are peroxideelectrodes. Peroxide is a product of a glucose oxidase catalyzedreaction. The peroxide electrodes can be used in conjunction with anoxygen reference which could be polarographic or optical or anysubstantially non-oxygen consuming probe. The depth of the interrogationcan be adjusted. Furthermore, in illustrative examples, the region isspatially sampled with more than one light path to provide greaterprecision in monitoring changes in the oxygen field of the reactionzone. Oxygen, glucose, or a product of the reaction of oxygen andglucose can be detected.

As a reversible oxygen binding protein such as hemoglobin is presentthroughout the reaction region 002, it can serve as both an oxygentransporter and an oxygen probe. In other words, both the firstreversible oxygen binding protein, an exemplary oxygen transporter, andthe second reversible oxygen binding protein, an exemplary oxygen probe,can be hemoglobin. Therefore, in certain aspects the oxygen probe is anoptically sensitive molecule, for example an engineered hemeprotein or aheme derivative, such as hemoglobin. In other aspects, the oxygen probeis an optical probe such as a dye, which depending on the specific dyeused, can consume oxygen. The optical means of measuring the oxygensaturation of hemoglobin are well established, allowing oxygenconcentration to be measured optically in a sensitive and selectivefashion. This provides measurements of oxygen concentration, through andif needed outside of the enzyme reaction zone. Accordingly, in certainillustrative examples, the probes emit and receive light at an oxygensensitive hemoglobin absorption wavelength, or an oxygen sensitiveabsorption wavelength of another reversible oxygen binding protein. Anoxygen sensitive hemoglobin absorption wavelength is a wavelengthbelonging to the subset of the wavelengths of the hemoglobin absorptionspectrum for which absorption is a function of hemoglobin oxygensaturation. Where hemoglobin is used as the reversible oxygen bindingprotein the detector probes emit and receive light at between 600 and800 nm. Wavelengths greater that 800 nm also have the favorableproperties of low absorption and of changes in the absorption withoxygenation. Wavelengths shorter than 600 nm have very high absorptionand are less suitable for measuring oxygenation due to poor lightthroughput. For the device illustrated in the Examples herein, 635 nmwas chosen for its low cost and ubiquitousness in the manufacture andindustry of silicon chips, optoelectronics and integrated circuits. Twoother working wavelengths include, for example, 660 nm or 905 nm, whichare commonly used to measure hemoglobin oxygenation. A second wavelengthcan be used in addition to a primary wavelength to improve calibrationstability. In particular the isobestic wavelength of hemoglobin, 805 nm,can be used to provide an oxygenation independent measurement which iseffected by and provides a measurement of the amount of hemoglobinencountered in the light path.

The issues of selectivity are overcome by biosensing methods andbiosensor devices provided herein, when a background oxygen measurementis taken at a region in the glucose sensor that is distant from a site007 where glucose enters the sensor, but having an oxygen concentrationthat is similar to that initially present at the site where glucoseenters the device. Accordingly, to further deal with the issue ofsensitivity and dynamic sensing range, three surfaces 006, 007, and 008of the reaction region 002 are designed to create a glucose gradientalong the sensing boundary 008, which is also created by the very highlateral spatial normalization of oxygen of the oxygen transport region.Accordingly, at a point along a sensing boundary 008 in the reactionzone all of the measurable glucose has been converted to product, whileat the glucose entrance boundary 007, the glucose concentration ishigher. This design causes glucose to be drawn down a concentrationgradient from the glucose entrance boundary 007 towards the center ofthe reaction chamber. Consequently there is an oxygen gradient in thereaction chamber since it is consumed along with glucose by glucoseoxidase. When this oxygen field is spatially interrogated by the opticalsensing elements, the result is a profile that is dependent on the bulkglucose concentration. The result is that the enzymatic reaction remainsresponsive to changes in bulk glucose concentration over a broad rangeof bulk glucose concentrations, and this reaction couples bulk changesin glucose concentration to changes in the oxygen field in the reactionzone.

The composition of the reaction region 002 is designed to containsufficient glucose oxidase and reversibly oxygen binding protein so thatas glucose moves from the glucose inlet 007 through the reaction region002, its concentration is reduced such that at a point along thereaction region 002 away from the glucose inlet 007, the glucoseconcentration is reduced to a level that does not measurably reduce theoxygen concentration even in the presence of glucose oxidase. Thereference region 029 is the region within the reaction region 007 wherethe glucose concentration is sufficiently low so as not to measurablyreduce oxygen concentration in the reference region 029. The precisestart of the reference region 029 within the reaction region 002 dependson the reaction rate of glucose near the glucose inlet 007 which willsetup how deep the oxygen gradient extends. This is controlled by designvia glucose oxidase loading, reversible oxygen binding proteinconcentration, size of the glucose inlet 007, area of the reaction zone002, and the surface area ratio of the oxygen injector 006, to theglucose inlet 007. Therefore, the reference oxygen measurement aspect ofthe present invention is tunable and scalable. In certain aspects, theconcentration of an enzyme in the reaction region 002, and theconcentration of the reversible oxygen binding protein in the reactionregion 002 are tuned to provide an analyte sensitive oxygen gradientnear the inlet 007 such that the desired dynamic range of analyteconcentrations can be measured, and to provide a analyte insensitiveoxygen reference concentration distal to the inlet. The reference regionis at least large enough to encompass a region of the reaction region007 interrogated by one or more reference emitters and/or one or morereference detectors. A reference region 029 is located within thereaction region 002. The reference region 029 for example, can extend incertain aspects, from the center of the reaction region 007 with respectto a plane extending from the glucose inlet 007 to a distal boundary 027of the reference region, and can extend through the distal boundary 027.In certain aspects, for example, the reference region can begin about200 microns or so from the glucose inlet.

One or more reference detector probes 028, such as one or more referenceemitter fiber optic bundles, can emit light into the reference region029. For example, the reference probes can be located at least ½, ⅔, or⅘ the total length of the reaction zone from the glucose inlet. Incertain embodiments, the furthest 1, 2, 3, 4, or 5 fiber optic bundlesor adjacent pairs of fiber optic bundles, in an array of emitter fiberoptic bundles can be the reference emitter fiber optic bundle.Therefore, light can be emitted from one or more reference fiber opticbundles to excite an oxygen binding probe, such as hemoglobin, andabsorption or emission of light from the oxygen-binding probe can bedetected by one or more detector fiber optic bundles. This availabilityof the background measurement for oxygen within the reaction zone isuseful, as it provides a highly relevant background measurement for theoxygen availability to the enzyme without disturbing the reactionoccurring in the reaction zone. The reference measurement can be used todetermine absolute or relative analyte concentrations, and it canprovide a measure that can be used to map an oxygen profile in thereaction chamber

In certain illustrative embodiments the emitters 410 and/or thereceivers 415 are fiber optic fibers. In one implementation of thisdesign, the sensing boundary 008 is the end of a fiber optic bundle. Theresulting surface satisfies the requirement of a no flux boundarycondition. The diameter of the fiber optic bundle can be, for example,between 100 um and 300 um. In certain aspects, the receiver, sometimesreferred to as a receiver optrode, or the emitter, sometimes referred toas an emitter optrode, is a shaped fiber optic fiber. Typically, thesensor includes at least 2 receivers and/or emitters. This allows thespatial sampling of the reaction zone 002 disclosed herein, which can beperformed using more than one receiver, more than one emitter, or morethan one receiver and emitter. In certain aspects, the receiver is aminiaturized electronic optical receiver. The detector probes, such asthe fiber optic fibers or fiber optic fiber bundles, can be coated witha bioadhesive molecule that increases the adhesion of biomolecules tothe fiber or fibers. For example, the detector probes can be coated withpolylysine. The bioadhesive molecule serves to increase the adhesion ofthe matrix of the reaction region 002 to the detector probe. In certainexamples, the emitter and for detector are miniaturized electronicshoused within a housing of the sensor, in which case no fiber optics areused. The physics of the measurement for these examples is the same aswhere fiber optics is used, but the method of interrogation couldchange, as will be understood. In one aspect, fused fiber bundles (alsocalled imaging conduits) can be used. For example, fused fiber bundlesthat have a 50 um outer diameter, but contain an array of fused fiberswithin it, each, for example, 10 um in diameter. In this aspect, thereaction region can be sampled with a resolution of 10 um or less.

In one illustrative glucose sensor that utilizes fiber optic fibers, abiocompatible hollow micro-pore membrane tube, such as a micro poroushollow fiber membrane 11 permeable to glucose surrounds the tip of theoptical fiber bundle and extends beyond the optical fiber bundle. Thehollow micropore fiber membrane 11 covers the glucose entrance boundary007 (i.e., glucose inlet). The reaction chamber 002 is then formed byplacing a mixture of glucose oxidase, bovine serum album, and glucose,and oxygenated hemoglobin inside the hollow micro fiber membrane 11 andon the surface of the optical fiber bundle 008. The mixture is filled toa thickness of 50 um and is then cross-linked using glutaraldehyde.Liquid medical grade silicone is then added to hemoglobin in a quantitysufficient to form a suspension that will fill the hollow micro fibermembrane 11 creating the oxygen transport region 001 and contacting theexposed surface of the reaction chamber 002 to form a boundary 006.

Sensing then occurs in this illustrative glucose sensor by directinglaser light through subregions of the optical fiber bundle tointerrogate the reaction chamber oxygen field by monitoring thehemoglobin oxygen saturation. This light is then reflected at the oxygeninjector 006 back through the optical fiber bundle and measured by aphotodiode.

In certain illustrative examples, such as the glucose sensor illustratedin FIG. 3, FIG. 4, and Example 2, an array of fiber optic fibers areused as the emitters 410 to emit light toward a ½ circular bundle offiber optic fibers, the receivers 415. The array of emitter fiber opticfibers 410 can be formed into a loop such that light emitting from theend of the emitter fiber optic fibers 410 travels in a second direction478 that is substantially opposite, or opposite, to light that entersthe emitter fiber optic fibers in a first direction 475 produced by alight source 490. Therefore, in these aspects the emitters and thereceivers can enter the glucose sensor 400 through a first end 482 ofthe glucose sensor 400. This design makes it much more convenient forconnecting the glucose sensor to a light source 490, photodetector 495,and/or signal processing unit 498. Furthermore, this design improves thesignal to noise ratio of the device over one relying on measurements ofscatter, reflection, absorption or other optical phenomena in whichlight is emitted and detected from/by the same bundle. Since emitterfiber optic fibers are easily aligned with receiver fiber optic fibersthe amount of light collected can be orders of magnitude greater. Theemitter fiber optic bundle and the receiver fiber optic bundle can be acircular bundle, ½ circular bundle, or a side-by-side array, but inillustrative examples the emitter bundle is a side-by-side array and thereceiver bundle is a circular or ½ circle fiber bundle.

In another embodiment, provided herein is a spectrometer comprising alight source, a photodetector, an emitter fiber optic fiber or anemitter fiber optic bundle of fibers, for emitting light from the lightsource to a detection region, and a receiver fiber optic fiber, or areceiver fiber optic bundle of fibers, for receiving light emitted bythe emitter fiber optic fiber and transmitting the light to thephotodetector, wherein the emitter fiber optic fiber forms a loop suchthat light emitting from the emitter fiber optic fiber travels in asecond direction that is substantially opposite, or opposite, to lightproduced by a light source that enters the emitter fiber optic fibers ina first direction. As will be understood, many of the teachings providedherein directed to the sensing region of a biosensor apply to thedetection region of the spectrometer of the present invention. In fact,it will be understood that a sensor according to the present inventionutilizes a spectrometer of the present invention for its detection andanalyte measuring functions. For example, in certain aspects of thespectrometer, as well as in aspects of a biosensor of the presentinvention, such as a glucose sensor, the emitter fiber optic fiber andthe receiver fiber optic fiber can enter the microspectrometer through afirst end. In a related embodiment, provided herein is a method foroptically analyzing a sample by transmitting light from a light sourceinto an emitter fiber optic fiber through a sample into a receiver fiberoptic fiber in optical communication with the emitter fiber optic fiber,wherein light travels from the light source through a loop in theemitter fiber optic fiber, or bundle of fiber optic fibers, such thatlight exiting from the emitter fiber optic fibers travels in a seconddirection that is substantially opposite, or opposite, to light producedby a light source that enters the emitter fiber optic fibers in a firstdirection. Therefore, in these aspects light can enter and exit thespectrometer from a first end, which combined with the small size of thedevice, simplifies and improves the possible uses for a spectrometerprovided herein.

Virtually any light source and photodetector can be used for thespectrometer or sensor of the present invention, such as a glucosesensor. The light source can be broad or narrow banded in the lightspectrum and can be from a selection of multiple bands available to theemitter fiber optic fiber(s). In certain aspects, the light source is alaser light source, an LED, an incandescent light bulb, an arc lamp, andin illustrative embodiments the light source is a laser diode. Theemitter fiber optics fibers can be connected internal or external opticsor optoelectronics to control the wavelength, intensity, polarization,pulse width, or other optical characteristics of the light. The receiverfiber can be connected to additional internal or external optics oroptoelectronics for measuring intensity, polarization, fluorescent decayor other optical properties in narrow or wide bands of the lightspectrum. It will be understood that although the invention isillustrated herein by the use of a light source and a photodetector,other types of energy sources and detectors, respectively, can be used.

In an alternative sensing scheme, an oxygen sensitive dye can be placedat the sensing boundary 008 of a biosensor or spectrometer providedherein. The dye can then be probed optically to monitor the oxygen fieldand assess the glucose concentration.

The detector 495, light source 490, and signal processing unit 498 areconnected to one end of the biosensor or spectrometer provided herein.In certain illustrative examples, the detector 495, light source 490,and signal processing unit 498 are housed within the same assembly. Forglucose sensors provided herein, typically probes of the sensors, orthin tubes or wires or other coverings that house the probes, passthrough the skin and connect to a housing outside the skin that includesthe detector 495, light source 490, signal processing unit 498 and inthe case of internalized electronics, supporting electronics.Accordingly, in these illustrative examples, the device is a transdermaldevice. In certain aspects of a transdermal device a non-disposable unitexternal to the skin is connectorized to connect with a disposableimplantable needle-type sensor connectorized on its back end. In such ascenario, the two units are connected together such that, in oneillustrative example, light sources and photodetectors within thenondisposable unit are aligned, via the connection, with light guideshoused in the disposable needle-type sensor which terminateappropriately near or adjacent to the glucose reaction zone within thedisposable implant. The nondisposable unit manages timing ofmeasurements, data analysis, power supply, communication with thepatient, etc. . . . which the disposable unit houses the sensor probe.

In certain illustrative aspects, as illustrated in Example 2, spatialsampling of the reaction region is achieved by turning on certainemitter fibers at different times. For example, individual emitterfibers 412, adjacent pairs of emitter fibers, or adjacent trios offibers within an emitter array 410 can be turned on and off in orderfrom closest or furthest from the glucose inlet 7, to the opposite end.In these aspects, a single photodetector 495 can be used to measurelight at each time point that a pair of emitter arrays 410 is turned on.These measurements can be used to recreate the spatial oxygen andglucose content of the reaction region 002, and provide a substantialincrease in signal to noise ratio over aspects where all emitter fibersare turned on simultaneously.

In certain aspects, because the reduction of glucose results inperoxide, the surrounding tissues can be protected from any suchsecretions from the sensor. This can be accomplished by reducing theperoxide to water and oxygen in the space immediately surrounding thereaction chamber via the enzymatic reaction of peroxide with catalase.

To prevent the drift in the oxygen sensing process, agents (e.g.catalase, dehydrogenases) can be added to prevent any free radicals thatare created during the optical interrogation from degrading thehemoglobin emulsion. One such agent is blood serum albumin, which canalso be used as soluble carrier proteins for crosslinking to the glucoseoxidase. The glucose oxidase enzyme is crosslinked to the bovine serumalbumin to stabilize enzyme activity over the period of monitoring. Incertain aspects the soluble carrier protein may be bovine serum albumin,human serum albumin, and/or gelatin. At high glucose oxidaseconcentrations (on the order of 70% or greater) it may be unnecessary toadd the soluble carrier proteins to form a hydrogel (See e.g., Clark,U.S. Pat. No. 6,815,186). As a general method according to the presentinvention, initial albumin concentrations from 15 g/L to 25 g/L can beused in formation of the matrix or hydrogel. This value is based on therange of normal human albumen which is 35-47 g/L, divided by two(assuming a hematocrit of 50%) (data from the worldwide web athoslink.com/LabResults/refranges.htm).

Glucose sensors provided herein can be calibrated before implantation byexposing them to bulk oxygen concentrations from 3% to 7% and fromglucose concentrations from 30 mg/dl to 500 mg/dl. The individualprofiles monitored are then used to precisely quantitate unknown glucosemeasurements in the presence of variable bulk oxygen tensions.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Analysis of the Oxygen Conducting Capability of Hemogloblin

The following example illustrates the oxygen conducting capability ofhemoglobin and the intended functionality of the reversible oxygenbinding protein. A film of hemoglobin was prepared as follows. Humanblood was extracted with a finger-stick device. A large drop of theblood was deposited on one end of a rectangular microscope glass slide.A blood smear ⅛ inches by 1½ inches in dimension was created by slidinga microscope cover glass from the blood drop, across the glass slide tocreate a thin film of blood across the glass slide. The glass slide wasthen covered in cellophane leaving approximately ¼ inch of the bloodsmear exposed in the long axis. A glass coverslip was placed on thecellophane to ensure a good seal. This setup ensures no-flux oxygenboundaries on all surfaces and edges of the blood smear, except for atthe exposed ¼ inch at the end. A diode laser's beam was directed throughthe glass slide, blood smear, cellophane, and cover glass at a point 1inch from the exposed boundary. Light leaving the sample was measured bya photodiode. The photodiode voltage, which is proportional to lightcollected was monitored over time before, during and following changingoxygen concentrations at the exposed boundary.

The signal in FIG. 2 represents the changing Hemoglobin absorption at635 nm laser light. This figure shows the photodiode response first forthe transition from room air at the boundary to a 5% oxygen, 95%nitrogen gas mixture (arrow A), and then for the transition from thatgas mixture back to room air (arrow B). As can be seen, the photodiodevoltage changes only seconds after the boundary condition changes.Therefore a significant amount of oxygen moved 1 inch within seconds, arate far beyond that expected by diffusion. This experiment isfundamentally identical to the oxygen transport scenario within thesensor implanted in the body; the oxygen transport region will beproximal to the high oxygen tension arterioles and capillaries and willbe coupled to the reaction zone where oxygen will be consumed. Thisexperiment demonstrates that the oxygen from the body can be rapidlydirected to the site of the enzymatic reaction where it is needed.

Example 2 Illustrative Implantable Glucose Sensor

This Example discloses certain features of an illustrative glucosesensor 400 of the present invention, as illustrated in FIG. 3 and FIG.4, and exemplifies a method for manufacturing the illustrative glucosesensor 400. In the illustrative glucose sensor 400 provided in thisexample and illustrated in FIG. 3, the electronic light source 490 andthe photodetector 495 communicate through a series of fiber opticbundles 410, 415 with the illustrative glucose sensor 400. In the methodused to create the glucose sensor 400, an array of individual opticalfiber emitters 410 was bent into a hook so that light emanating from theemitter array 410 traveled through the reaction zone 402 and wascollected by a detection fiber optic bundle 415 (i.e. an array ofoptical fiber receivers). In this illustrative glucose sensor 400 theentire back end of the detection bundle 415 was coupled to a photodiodetype photodetector 495.

During use of the glucose sensor 400, spatial sampling of the reactionzone 402 was accomplished by changing the pattern of emission down theemission array 410. In one exemplary method, adjacent pairs of emitterfibers within the array 410 were turned on in order from closest to theglucose entry boundary (i.e. glucose inlet) 407 inwards towards theoxygen reference region where the oxygen reference region is at least asfar from the glucose inlet as the distance, in a direction pointingalong the surface normal of the glucose inlet, at which oxygenconcentration changes are glucose independent, each time recording theoutput of the single photodiode. The oxygen reference region was theregion within the glucose reaction region 402, running between 200 umfrom the glucose inlet to the border 427 of the glucose reaction regionopposite the glucose inlet 407. This border region 427 opposite theglucose reaction region in the illustrative glucose sensor wasapproximately 300 um from the glucose inlet 407. By knowing the emissionsource corresponding to each output result, the spatial content of thereaction zone can be reconstructed. A key advantage to this mode ofoperation over that in which all emission fibers are on simultaneouslyand the detection bundle is broken into subsets, each with its ownphotodetector 495, is the substantial increase of the signal over thenoise. Details of the method that was used to make the illustrativeglucose sensor 400 are provided in the following paragraphs.

The Implant Needle 480

Implant needles 480 were custom build by modifying commerciallyavailable 23 gauge syringe needles. A window 470 was carved at a pointon the side or wall of the needle between the tip 481 of the needle 480and the back end 482 of the needle 480 using a high speed rotary toolwith a ceramic tip. The window 470 was cut 1 mm long, and spanned about½ the curvature or circumference of the needle 480. The tip of therotary tool was chosen to smooth the boundary of the window between theouter and inner wall of the needle while the rotary tool cuts. The goalof the smoothing was to minimize surface tension boundaries, and tofacilitate the entry of tissue into the space between the inner andouter walls of the window's boundary to minimize diffusional distancesto the reaction zone

The Emission Array 410

The emission array 410 was a linear array of individual fibers 412 witha cladding diameter (i.e., an outer diameter) of either 40 μm or 50 μmand a numerical aperture of 0.54, 0.57, or 0.64. The fibers 412 had anumerical aperture of 0.54. The total length of the array 410 wasslightly smaller than the inner diameter of the implant needle 480. Forthis illustrative example, that corresponded to approximately 10 fibers.It was found that when coupling emission fibers 412 to the primary laseroutput fiber coupler using a multimode FC connector with a 125 μm bore,pairs of fibers couple as efficiently as triplets. Additionally singlefibers 412 coupled poorly, likely as a result of bending about theoptical axis due to stresses in the fiber 412, and the extra room in theconnector. Triplets of fibers do not fit linearly within the coupler,and sample a larger volume of the reaction zone. As such, we chose tocouple pairs of individual fibers 412 within the emission array 410,each to their own laser source.

A notable feature of this illustrative glucose sensor 400 is the 180degree bend in the emitter array 410 allowing the emitter array 410 toenter the back 482 of the implant needle 480, to pass the reactionwindow 470 carved in the needle 480, and then to turn around in theneedle 480 and align in a parallel orientation adjacent to the detectionbundle, also referred to herein as the receiver bundle 415, asillustrated in FIG. 3. Thus, the emission bundle 410 and the detectionbundle 415, run parallel for a stretch of the illustrative glucosesensor 400 at the back end 482 where the emission bundle 410 and thedetector bundle 415 enter the glucose sensor 400. The bend 414 in asingle fiber 412 is formed by inserting it into a 30 gauge needle toform a loop and reducing the size of the loop until just slightly largerthan its breaking point. A heating source, in this case a cigarettelighter flame was applied momentarily to melt the fiber and cause it toyield to the applied stresses induced by the bending. Likewise a set offibers can be bent as a group by feeding them in to a 28 gauge needleand forming a loop slightly larger than the size at which they break.Again, a cigarette lighter flame was applied briefly to the fibers tocause them to yield to the stresses applied by the loop. In both cases,if the resulting loop is too large in its bending radius, the processmay be repeated by further retracting the loop into the needle to placeadditional stress on the formed loop and momentarily heating the loopwith the flame from a cigarette lighter, actually a Quicker Clicker™lighter for a fireplace log. After testing the flame from a variety ofsources, including a butane pencil torch, a match, a propane torch, anda map torch, it was found that only a very small flame was needed, andthat too large of a flame caused unintended bending out of the intendedplane of the loop. The area of the emission array is small enoughcompared to the overall area of the glucose sensor and the overallvolume within the lumen of the needle such that an oxygen transportregion 401 can surround the emission array in the lumen of the needle480 beyond the reaction window 470 and still function as originallyintended.

The Detection Bundle 415 (Also Referred to Herein as a Receiver Bundle)

The detection bundle 415 in the illustrative example is a circularbundle of individual fibers 416, in which one half of the circularcross-section is removed. That is, in cross-section the bundle forms ½ acircle. The geometry facilitates hand manufacturing at a high throughputrate. For large volume fabrication, alternate geometries can be used.Bundles were constructed two at a time by constructing a circularbundle, and then splitting the bundle into two semicircularcross-sectional bundles, each used in its own device. The circularbundles were constructed by filling a smaller gauge needle (28 gauge)with as many individual fibers as possible. 50 μm fibers as describedabove in the emission array 410 section were used. The individual fibers416 are then held together using nail polish according to the followingprotocol. The individual fibers 416 are threaded into the needle fromthe pointed end 481 of the needle 480, and out the blunt back end 482 byat least one inch. A generous amount of nail polish is deposited at theback end 482 of the needle 480, held in place by the surface tension ofthe needle/fiber/polish interface. The bundle 415 is then drawn backinto the needle, past the blunt end 482, and then advanced again so theend of the bundle 415 aligns with the blunt end of the needle. At a timebefore curing of the nail polish is complete, the bundle 415 is againadvanced out of the needle 480, allowing the fibers 416 to cure but notbe adhered to the needle 480. Before curing is complete, the bundle 415is carefully split in half with a sharp Exacto™ knife blade, along adiameter of the cross-section and allowed to cure. The end result is twohalf-circular cross-sectional bundles.

Depositing the Reaction Region 402 onto the Detection Bundle 415

In this illustrative glucose sensor, the reaction region (also calledthe reaction zone) 402 is deposited onto the detection bundle surface408 outlined below.

1. The detection bundle pair was either reinserted into the pointed endof a 28 gauge needle or remained in the 28 gauge needle used to form thebundle pair, and aligned with the blunt back end 482.

2. The end of the detection bundle 415 and the emitter bundle 410 waspolished to increase light collection efficiency. We have found that anincrease in light collection efficiency of approximately 3× can beachieved by polishing the collecting end of the detection bundle 415 andthe emitting end of the emitter bundle 410. Polishing was achieved firstby slightly advancing the bundle beyond the blunt end 482 of the 28gauge needle and then by gliding the bundle end on a series of polishingpapers of increasing fineness as using known techniques to polish fibersby hand. “Thor's Guide to Connectorization and Polishing Optical Fibers”Thor Labs, Newton, N.J., 1997.

3. Fiber bundle tip was dipped in solution of 0.1% (w/v), orequivalently 1 μg/ml, poly-lysine in deionized water. The poly-1-lysinewas used to facilitate the adhesion of biomolecules to the end of thedetection bundle 415. Polished glass has poor adhesion to biomolecules.

4. The prepared Reaction region mixture is deposited onto tip of bundle408. The reaction mixture is a stabilized matrix comprised ofhemoglobin, an engineered hemeprotein or a heme derivative such asmyoglobin, the enzyme glucose oxidase, a carrier protein such asalbumin, and a fixative such as glutaraldehyde This is accomplished byfirst drawing the bundle 415 back into the 28 gauge needle approximately2 mm from the blunt end of the needle. The blunt end of the needle isused to cut the reaction zone matrix, which has been deposited onto aflat surface such a glass slide, into the exact shape of the needle'sinner lumen, analogous to a cookie cutter. Two variations were used toaccomplish this step

a. An entire circular bundle 415 was used, and split into two bundlesafter depositing the reaction zone

b. A pair of half-circle bundles 415 were used

5 The bundle 415 is carefully pushed back though the blunt end of the 28gauge needle, carrying the cut reaction zone 402 with it.

6. The needle is removed from the bundle 415, and the bundle 415 isdipped into a silicon adhesive diluted with Toluene at a ratio of 6:1Toluene to adhesive, to coat the bundle tip and reaction zone 402 withan oxygen permeable, glucose impermeable membrane.

Assembly of the Illustrative Glucose Sensor 400

The illustrative glucose sensor 400 was assembled by aligning theemission array 410 with the detection array 415 outside of the implantneedle 480 such that the reaction region 402 is between the emissionarray 410 and the detection array 415, and then sliding the alignedemission array 410, reaction region 402, and detection array 415 (i.e.the coupled light system) back into the needle 480 such that thecoupling region (i.e. the region between the emission array 410 and thedetection array 415, which includes the glucose reaction zone and inwhich light from the emission array 410 is received by the detectionbundle 415) is within the reaction window 470 cut into the needle 480.The steps are outlined below in more detail.

1. The detection bundle 415 and the emitter bundle 410 are both eitheradvanced up through the implant needle 480, or backed down through theneedle 480 so that a few inches of both extend beyond the pointed end481 of the needle 480, including the bend 414, and emitting end 413 ofthe emitter bundle 410 and the receiving end 408 of the detection bundle415, and length of the detection bundle 415 and the emitter bundle 410extends well beyond the back end of the implant needle.

2. The emitting end 413 of the emission bundle 410 is positioned nearthe receiving end 408 of the detection bundle 415 until maximumtransmission is achieved through the back end of the detection bundle415. This position will correspond to a geometry in which the lineararray of the emission bundle 410 is aligned transversely to the linearboundary of the half-circular bundle. That same boundary, along with thelinear shape of the emission bundle 410 remove rotational degrees offreedom from the alignment procedure. Ideally, the system is constructedso only the axial placement is critical, but some transverse translationof the emitter bundle 410 relative to the detector bundle 415 may berequired.

3. An adhesive such as silicon or nail polish is applied along thelongitudinal coupling of the emission bundle 410 and detection bundle415 behind the optical coupled region. This step stably locks the twofiber systems together.

4. The coupled light system is then backed into the needle 480 so thatthe coupling is within the reaction window 470 cut into the needle 480.

5. The length of the needle 480 between the reaction window 470 and thetip 481 of the needle is filled with the oxygen transport region 401material and allowed to dry. The oxygen transport region is a stabilizedmatrix comprised of hemoglobin, an engineered hemeprotein or a hemederivative such as myoglobin, a carrier protein such as albumin and afixative such as glutaraldehyde. The mixture is applied to the sensorbefore its cure time has completed such that it still behaves as aliquid.

6. A glucose entry pinhole 407 is cut into the side of the reaction zone402 using a the pointed tip of a small syringe needle to allow glucoseto enter into the reaction zone.

7. The reaction window 470 is covered with a glucose and oxygenpermeable membrane.

The entire needle 480, or only the reaction window region 470 is coveredwith a biocompatible membrane. For example, the membrane can be amembrane with approximately 5 μm pores to promote immune reactionresistance.

Example 3 Glucose Measurements Using an Illustrative Sensor

This Example demonstrates detection of glucose in a relatively simpleglucose sensor that includes two stabilized oxygen transport matrices incommunication with one another. One of the illustrative stabilizedoxygen transport matrices was an oxygen transport region and the otherwas a glucose reaction region that included a stabilized glucoseoxidase-hemoglobin thin gel.

A contiguous oxygen transport region and glucose reaction region weredeposited on a glass slide, coated with silicon, and a glucose inlet waspresent on a surface of the glucose reaction region. The glucosereaction region was made with 20 mg glucose oxidase and 0.1 ml of humanblood crosslinked with a dilute formaldehyde solution. The steps forconstructing the glucose reaction region are outlined below in moredetail.

1. 0.1 ml of whole human blood was combined with 0.3 ml of a solutioncontaining a 1:3 ratio of 70% isopropyl alcohol to distilled water tolyse the red blood cell membranes.

2. 20 mg glucose oxidase was added to the lysed blood cell mixture ofstep 1. The mixture contained no visible glucose oxidase precipitatefollowing adequate mixing.

3. Using the edge of a glass coverslip, a portion of the mixture of step2 was transferred to the bottom portion a glass slide to form a thinstrip across short dimension of the slide. The width of the strip was 2mm.

The mixture was allowed to dry on the glass slide.

The glass slide was immersed in a 4% formaldehyde solution for 10minutes and then rinsed with 70% isopropyl alcohol and allowed to dry

The oxygen transport region was made with 0.1 ml of human bloodcrosslinked with a dilute formaldehyde solution. The steps forconstructing the oxygen transport reaction region and completing thesensor are outlined below in more detail.

1. 0.1 ml whole human blood was combined at a 1:3 ratio to a solutioncontaining a 1:3 ratio of 70% isopropyl alcohol to distilled water tolyse the red blood cell membranes

2. The mixture was mixed to a uniform consistency and about ½ of it wasapplied to the glass slide containing the glucose reaction region sothat the mixture covered the majority of the glass slide across itsshort dimension and formed a contiguous layer with the glucose reactionregion. The mixture was allowed to dry.

3. The entire preparation on the glass slide was covered with a thinlayer of silicon rubber. Using a very sharp blade, parallel grooves werecut through the silicon rubber and the deposited layers. A sensor isthus defined by the contiguous regions between two parallel grooves. Thesilicon was blow dried with cool air to accelerate curing.

4. Using the tip of a syringe needle, a glucose inlet was cut throughthe silicon at the intersection of one of the grooves and the glucosereaction region just below where the oxygen transport region and theglucose reaction region connect.

The glass slide was suspended in a water bath with a [5% O₂+95% N₂] gasmixture bubbled in. The reaction zone was optically probed by focusedlaser light in a spot approximately 10 um in diameter positioned 50 umor 100 um from the glucose inlet along the axis of the injector surface.Laser light was recollected onto a photodiode whose electrical currentwas converted to voltage and recorded across a clinical range of glucosesolutions in the bath. FIG. 6 shows graphs of glucose concentrationswithin the bath plotted against the corresponding photodiode voltagewhen the reaction gel was interrogated at a location of 50 um or 100 umfrom the entrance for glucose (i.e. the glucose inlet). The graphsdemonstrate the utility of spatial sampling within the reaction zone.The interrogation spot 50 um in from the glucose inlet demonstrated goodsensitivity to glucose concentration changes at lower glucoseconcentrations, but looses sensitivity near 150 mg/dl at which point thepower function relationship transitioned into steepness. Theinterrogation spot located 100 um in from the glucose inlet demonstratedgood sensitivity at 150 mg/dl up to at least 300 mg/dl. Information fromthe two locations together yields good sensitivity across the clinicalrange of glucose concentrations. Glucose solutions were measured intriplicate using a TrueTrack™ over-the-counter glucose meter co-brandedby Say-On/Osco/Albertsons and Home Diagnostics, Inc.

FIG. 7 shows the calibrated response of the glucose oxidase-hemoglobinthin gel of FIG. 6 plotted against the TrueTrack™ glucose metermeasuring the same glucose solutions. A linear fit yields an R² value of0.99, thus demonstrating that results obtained using the illustrative.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1-55. (canceled)
 56. A sensor for measuring an analyte, the sensorcomprising: an oxygen transport region comprising at least onereversible oxygen binding protein; an analyte reaction region, theanalyte reaction region comprising: an oxygen-permeable surface incommunication with the oxygen transport region; an analyte-permeablesurface configured to allow a target analyte to pass through, theanalyte-permeable surface located generally orthogonal to theoxygen-permeable surface; and an analyte reaction chamber at leastpartially surrounded by the oxygen-permeable surface and theanalyte-permeable surface, the reaction chamber configured to retain atarget analyte oxidase enzyme; wherein the oxygen-permeable surface, theanalyte-permeable surface, and the analyte reaction chamber areconfigured to form an oxygen gradient in the analyte reaction region;and a sensing region comprising at least one detector, wherein thedetector is configured to spatially sample the oxygen gradient withinthe analyte reaction region.
 57. The sensor of claim 56, wherein theanalyte-permeable surface is smaller than the oxygen-permeable surface.58. The sensor of claim 56, wherein the sensor is substantiallynon-oxygen consuming.
 59. The sensor of claim 56, wherein the oxygentransport region comprises a hemeprotein, or an engineered hemederivative.
 60. The sensor of claim 56, wherein the target analyte isselected from the group consisting of glucose, galactose, lactose,peroxide, cholesterol, amino acids, alcohol, lactic acid and mixturesthereof.
 61. A sensor for measuring an analyte, the sensor comprising:an oxygen transport region comprising at least one reversible oxygenbinding protein; an analyte reaction region containing a target analyteoxidase enzyme in communication with the oxygen transport region,wherein the reaction region is configured to allow a target analyte topass through an analyte permeable surface from a first portion to asecond portion, oxygen in the analyte reaction region interacting withthe analyte and forming an oxygen gradient from the first portion to thesecond portion; and a sensing region comprising at least one detector,the sensing region configured to measure the oxygen gradient.
 62. Thesensor of claim 61, wherein the oxygen gradient has more oxygen near thefirst portion than the second portion.
 63. The sensor of claim 61,wherein the detector spatially interrogates the oxygen gradient.
 64. Thesensor of claim 61, wherein the oxygen gradient is dependent on analyteand target analyte oxidase levels.
 65. The sensor of claim 61, whereinthe analyte reaction region further comprises a reference region,wherein analyte concentration in the reference region is sufficientlylow so as not to measurably reduce oxygen concentration in the referenceregion.
 66. The sensor of claim 65, wherein the reference region islocated after the second portion, so that the second portion is locatedbetween the first portion and the reference region.
 67. The sensor ofclaim 65, wherein a size of the reference region depends on a reactionrate of glucose near the first portion.
 68. The sensor of claim 65,wherein the reference region provides an analyte insensitive oxygenreference concentration.
 69. The sensor of claim 65, wherein the atleast one detector is configured to measure oxygen concentration in thereference region.
 70. The sensor of claim 61, wherein the at least onereversible oxygen binding protein comprises a hemeprotein, or anengineered heme derivative.
 71. The sensor of claim 61, wherein thetarget analyte is selected from the group consisting of glucose,galactose, lactose, peroxide, cholesterol, amino acids, alcohol, lacticacid and mixtures thereof.
 72. A method for measuring an analyte, thesensor comprising: providing a target analyte to an analyte reactionregion containing at least one target analyte oxidase enzyme, whereinthe target analyte progresses from an analyte permeable surface to adistal end of the analyte reaction region; transporting oxygen in anoxygen transport region to the analyte reaction region using at leastone reversible oxygen binding protein, wherein the oxygen enters theanalyte reaction region in a direction generally orthogonal to thedirection of the target analyte progression; reacting the transportedoxygen with the target analyte and the target analyte oxidase enzyme inthe analyte reaction region; and measuring the oxygen gradient in asensing region.
 73. The method of claim 72, wherein measuring the oxygengradient comprises spatially interrogating the oxygen gradient.
 74. Themethod of claim 72, wherein the target analyte is selected from thegroup consisting of glucose, galactose, lactose, peroxide, cholesterol,amino acids, alcohol, lactic acid and mixtures thereof.
 75. The methodof claim 72, wherein the measuring is performed at least at a first timepoint and a second time point, thereby providing information regardingchanges in oxygen concentration.